Title: DNA Microarraybased test systems:
1DNA Microarray-based test systems
- Achieving High Quality Data
Dr. Roland Toder r.toder.consulting Germany
2Biochips or arrays with various biological
material as tagged probes
Array formats are divided on the basis of feature
density. Macroarrays have less than 500
features/sqcm microarrays have more than 500
features/sqcm.
3- Pharma
- Pharmacogenetics (pharmacokinetic) personalized
medicine (haplotyping) - Human Genetics
- neonatal screening
- Infectious disease
- subtyping of pathogens
- Transplantation medicine
- HLA-typing haplotyping - homo-/heterozygotes)
- Cancer
- evaluation of aggressiveness part. tumors
- Pharma
- Elimination of drug candidates likely to produce
unacceptable side effects. - Proteins and their operation in biological
pathways of disease and enlist them as targets
for the development of new and better medicines - pharmacodynamics (amount of metabolizing enzymes)
- Cancer
- subtyping of cancers and stage (B-cell lymphoma)
- cancer treatment (chemotherapy metastasis)
- Infection
- stage of disease (infection) depending on the
enzyme-cascade
Infectious Disease Pathogen screening
(sepsis-chip) Blood bank
4Expression profiling approach in Diagnostics
- First step Broad approach (many genes) in order
to identify relevant genes - this is the most critical step where the quality
of the data is important. - Example
- Screening of women which have had breast cancer
in order to find out whether they do need
systemic drug therapy to prevent tumor spread
(metastasis) after surgery. - 100 women where screened with a chip containing
25,000 genes - A70 gene signature was sufficient to indicate
whether metastasis do appear soon.
5SNP Detection
- By creating microarrays that detect
disease-associated DNA sequence changes
(polymorphisms or mutations), this technology may
be used to identify groups of DNA sequence
changes that predispose patients to certain
diseases.
Human variation at the level of single nucleotide
polymorphisms may hold the key to effective
disease treatment.
6Examples in medical DNA diagnostics
HLA typing Chip from Genescan Europe AG
- DQA1 of the chip as an example
- HLA-DQA1 22 alleles
- Sequence-specific oligos out of 13 polymorphic
regions of exon 2 and 3 of the HLA-DQA1-gene on
the chip combination of 3 oligonucleotides per
allele allow the typing of the 22 alleles
7Allel 01021
controls
a b c
Oligo variants
1 2 3 4 5 7 8
9 11 12 13 14 15
Cy5-control
a b c
Perfect match
1 mismatch
8Allel 0303
controls
a b c
Oligo variants
1 2 3 4 5 7 8
9 11 12 13 14 15
Cy5-control
a b c
Perfect match
1 mismatch
9NOC-PCR (Genescan Europe AG)
One Amplicon multiple internal solid phase primer
HLA - DQA haplotypes
10HLA/DQA Genotyping with NOC-PCR
DQA 102/05
DQA 201/05
11HLA/DQA Exon 2 comp. 0201/05-0102/05
1,2
1
0,8
T
0,6
mA
A
0,4
0,2
0
0102/05
0201/05
Genotype
12HLA/DQA Exon 2 comp. 0201/05-0102/05
13Liver-Chip (GeneScan Europe AG)
C
A
B
(Yellow-Experiment) HepG2-RNA-Cy5 HepG2-RNA-Cy3
HepG2-RNA Cy-5 Jurkat-RNA Cy-3
14Using Microarray Data for Drug Discovery What
is necessary?
- Standards and Controls
- More stringent requirements for process control
and methodology needed - Measurable repeatability
- Measurable reproducibility
- Process definition
- Quality metrics
- Process must be robust to stand up to regulatory
scrutiny
15(No Transcript)
16Microarrays are a Closed Loop Process
Chip Chemistry
Statistical Analysis
Closed-loop Process
Probe Labeling
Detection
Standardized Kits Available
Hybridization and Washing
Standardized Kits Available
17What is necessary to obtain quality data in the
detection process?
- Right Resolution
- For proper sampling of spot data
- Signal to Noise Ratio
- To better discriminate between real data and
system noise - Repeatability
- For replicable results
- Statistical Analysis
- Proper metrics for spot quality, and for
determining data trends
18Internal controls for each spot will be necessary
for diagnostics
- A false positive or false negative result in
diagnostics can be fatal - So what degree of stability, security, and
safety of data acquisition should be provided
for an application biochip in medical
diagnostics? - (FDA/international standards)
The quality of data acquired through the analysis
system is therefore the most crucial and
critical step.
19Image Sampling Right Resolution
Simplest Case T-Test of means for 2 spots.
- Assumptions Normal distribution, 2-tailed test.
- Statistical sampling can be determined a priori
with a power analysis. - Power 1- ?
- Where ? is the probability of failing to reject
the null hypothesis when it is false. In other
words, failing to find a difference that actually
exists.
20Image Sampling Pixels/Spot
Effect Size and Power
90 ?m diameter spot
5 ?m
10 ?m
15 ?m
3.25 ?m
Large gt 99.9 Medium 99 Small 85
Large 95 Medium 60 Small 27
Large 58 Medium 27 Small 9
represents the smallest spots made due to
variation in contact printing
Cohen, J. (1988) Statistical power analysis
(2nd ed.). Hillsdale, N.J. Erlbaum.
21Repeatability and Reproducibility
- Reproducible and predictable performance of a
system is essential for providing the best data
and process control. - Typical laser scanners produce R2 values from.60
to .98 - CCD
- Sampling statistics
- Constant Gain of the detector
- R2 values from .95 to .98
- slide to slide
Meaning 95 of the data is attributable to the
experimental conditions and only 5 must be
determined with repeated scans.
22Reproducible Systems
Chip Chemistry
Statistical Analysis
Closed-loop Process
Probe Labeling
Detection
Hybridization and Washing
23Quality Data - Signal to Noise (SNR)
- Critical for detecting small changes in signal or
detecting weak signals - Noise in the acquisition system makes
determination of significant data at low levels
difficult. - Background subtraction methods affect the
linearity of the data
Low level gene expression is difficult to
determine unless system has low noise and high
data integrity.
24SNR is the foundation of image quality.
Defined as the mean of the signal divided by its
standard deviation.
25CCD Integration Time Yields More Photons
- A CCD device can detect approximately 400x more
photons than a laser scanner. - Simultaneously sampling 200,000 pixels with the
CCD allows a much longer collection time, and
therefore many more photons/pixel are detected. - This greater number of photons yields a high
signal-to-noise ratio (SNR), which is a key
determinant of a high spot and image quality.
26SNR Test Repeat Scan Comparison
Laser Scanner
arrayWoRxe
Ratio of Cy3/Cy3 0.998 ?0.4569 (CV 0.46)
Ratio of Cy3/Cy3 0.987 ?0.0321 (CV 0.03)
- This test reveals scanner statistical noise
independent of intensity differences on the
slide. - Same slide scanned 2x on each scanner.
- Same area compared from each scan (background, no
spots). - Overlay each replicate wavelength and measure the
pixel ratio coefficient of variation (CV). - There is good agreement between the predicted and
actual measurement accuracy for both scanners.
27Data Quality Coefficient of Variation of the
Ratios (CVR)
- The CVR is a collapsed measure of both the
quality of the chemistry and the quality of the
image. - Statistical analysis of spot intensity gives a
measurement of the relative expression of the
gene (the ratio between the 2 probes used in the
experiment) but also the confidence in the
measurement. - The CVR is the normalized variance for the spot
ratio, expressed as percentage of the ratio.
28Coefficient of Variation of the Ratios (CVR)
Defined as the standard deviation of the pixel
ratios of the two channels divided by the mean
pixel ratio of the two channels.
A low CVR value indicates high confidence in the
ratio.
29Coefficient of Variation of the RatioCVR
CVR is a Powerful metric that completes the
Closed-loop Process
CVR VariabilityProcess VariabilityDetection
Surface Chemistry Buffers Labeling Probe
Chemistry Humidity Spotting
Resolution SNR Geometry Uniformity Repeatability C
hannel Alignment
Fixing one without the other does not reduce the
CVR
30Coefficient of Variation of the RatioCVR
- Strong indicator of spot quality
- Strong indicator of experiment quality
- Can be used to weight spot data for inclusion in
bioinformatics analysis - With proper imaging, the ratio variability within
each spot is predictive of the variability
between experiments - Can be used to reduce the need for experimental
repeats
This tool will be very important in routine
analysis where a maximum reliability of each
array is required.
Brown, et al. (PNAS Vol. 98 no. 16
pp.8944-8949 2001)
31Conclusions
- DNA-arrays when used in diagnostics and pharma
have to meet a high degree of reproducibility,
reliability and quality in order to become a
standard tool. - The production of chips and necessary consumables
such as labeling systems washing and detection
systems, scanners and software have to meet
established certification systems such as ISO
GMP and FDA.